CN104849222B - The micro-fluidic apparatus for measuring concentration of rotation dish-style and method based on photometric detection - Google Patents

Abstract

The invention discloses a rotating disk microfluidic concentration measurement device and method based on photometric detection. A rotating disk microfluidic chip, a photometric detection device bracket, and an AC servo motor are arranged inside a sealed dark room, and the output shaft of the AC servo motor is vertical to the motor axis. A horizontal rotating disk microfluidic chip is installed upward and coaxially. The rotating disk microfluidic chip is equipped with an N-level test solution interpenetration zone, and each level of test solution interpenetration zone is based on the center It is an arc shape in the center of the circle, and the spring-like test solution flow channel between the adjacent two-stage test solution interpenetration zones is connected and communicated; the rotating disc microfluidic chip is used in combination with the rotating dilution positioning technology to realize the effective positioning of the dilution concentration, thereby Quickly calculate the dilution ratio, and multiply the dilution ratio by the corresponding relationship between the known optical path and the optimal concentration to generate the absorbance to deduce the precise concentration of the liquid to be tested. The process of sample injection, detection, and concentration calculation are all automated.

Description

Rotating disk microfluidic concentration measurement device and method based on photometric detection

technical field

The invention relates to microfluidic photometric detection technology, in particular to a microfluidic mixed solution concentration measurement device and method based on photometric detection.

Background technique

Microfluidic photometric detection technology is currently the most widely applicable microfluidic detection method. However, the photometric detection cell of the microfluidic chip is often fixed, and its optical path cannot be changed arbitrarily according to the concentration of the liquid to be detected. However, according to the spectrophotometric error theory, the optimal absorbance A _OPT in the measurement is 1/ln10, and the corresponding optimal optical path length is 0.434 αC ₀ ( C ₀ is the concentration of the absorbing substance in the solution, and α is the absorbing coefficient of the absorbing substance) . Therefore, only when the concentration is compatible with the detection optical path, the photometric detection noise is the smallest, and the detection error is also the smallest.

At present, in order to reduce the photometric detection noise caused by the inadaptation of the optical path, the device disclosed in the Chinese Patent Application No. 201210109396.4 titled "Method and Device for Adaptive Adjustment of the Optical Path of a Cuvette for Absorbance Detection of COD" can adjust within the range of Accurately adjust to the required optical path value, and realize the automatic adjustment of the optical path of the contrast color dish. However, in the actual detection process, the concentration value of the substance to be detected is often not known in advance, so it is difficult to judge in advance the selection of the optimal optical path value. In addition, this device is only suitable for photometric detection on a traditional scale, and is often not suitable for microfluidic photometric detection systems that require a higher optical path.

Contents of the invention

The purpose of the present invention is to aim at the detection error caused by the fixed optical path existing in the current microfluidic photometric detection equipment, and propose a rotating disc microfluidic high-precision concentration measurement device and method based on photometric detection. The detection and concentration calculation process are all automated, and the operation is convenient. It can realize the effective positioning of the dilution concentration and quickly calculate the dilution ratio. The corresponding relationship between the known optical path and the optimal concentration of absorbance is multiplied by the dilution ratio to reverse the detection. the precise concentration of the liquid.

The technical scheme adopted by the rotating disk microfluidic high-precision concentration measuring device based on photometric detection in the present invention is: a sealed darkroom is provided, and a rotating disk microfluidic chip, a photometric detection device bracket, an AC servo motor, and a photometric detection device are arranged inside the sealed darkroom. The bottom end of the bracket is vertically fixed at the center of the sealed dark room near the left edge, and the AC servo motor is fixed at the center of the sealed dark room near the right edge. Fluidic chip, close to the center of the rotating disc microfluidic chip and set up an original test solution pool symmetrically with respect to the center. The rotating disc microfluidic chip is equipped with an N-level test solution interpenetration zone, 3≤N≤100 , each level of test solution interpenetration zone is a circular arc with the center of the rotating disc microfluidic chip as the center, and the two original test solution pools are respectively connected by direct current channels and connected to the innermost first level of test solution interpenetration The test solution interpenetration zone of the adjacent two stages is connected and communicated with the spring-shaped test solution flow channel, and the outermost and last stage test solution interpenetration zone is a mixed test solution measurement zone whose width is larger than that of the previous test solution interpenetration zone; Between the two ends of the mixed test solution measurement belt is a laser measurement positioning hole that penetrates up and down. The photometric detection device bracket is connected to the laser light source directly above the laser measurement positioning hole through the right end of the first bracket loading arm, and through the second bracket. The right end of the loading arm is connected to the photomultiplier tube directly below the laser measurement positioning hole; the laser light source and the photomultiplier tube are respectively connected to the light source control module located outside the sealed darkroom through wires, and the light source control module is connected to the computer through the signal conversion module; The servo motor is connected with the motor control module located outside the sealed dark room through the motor control line, and the motor control module is connected with the computer.

The technical scheme adopted by the rotating disc microfluidic high-precision concentration measurement method based on photometric detection in the present invention is as follows: follow the steps in sequence;

1) The diluent with a concentration of 0 is injected into one original test solution pool, and the test solution is injected into the other original test solution pool. The computer controls the rotating disc microfluidic core to rotate, and the two different test solutions are subjected to centrifugal force from two The original test solution pool flows out and mixes in the test solution interpenetration zone at all levels and the spring-shaped test solution flow channel. The computer measures the number of times C and the timer that directly irradiate the photomultiplier tube through the laser measurement positioning hole according to the light signal emitted by the laser light source. Calculate the real-time rotation speed V _now = C/t of the rotating disk microfluidic chip from the measured time t , and adjust the AC servo motor speed until the real-time rotation speed V _now stabilizes at the target speed V _set ;

2) When the time reaches T ₁ /2, record the light intensity value I delivered to the computer by the current signal conversion module, and calculate the absorbance here A ₀ = I ₀ - I , I ₀ is the original light intensity signal, T ₁ is the rotating disk The rotation cycle of the microfluidic chip, T ₁ =2π/ V _set , the computer detects multiple times and judges whether the value of absorbance A ₀ has changed, if there is a change, continue to detect, if it is stable, mix the test solution and measure the test solution in the belt Concentration diffusion has stabilized;

3) The computer sequentially calculates the absorbance A obtained from the first moment to the nth moment, and makes a difference with the optimal absorbance A _OPT successively, that is, A _OPT - A , and obtains the absorbance value A _d when the difference is the smallest and the period T ₁ The relative time t _d of the absorbance value A _d is recorded, and the average absorbance value A _a and the average time t _a are obtained by averaging multiple cycles. If the maximum value A _max of the absorbance A in each cycle T ₁ is less than A _OPT , the concentration of the mixed test solution is too low, and the concentration factor Q=2× A _OPT / A _max is calculated according to the difference between the maximum absorbance A _max and the optimum absorbance A _OPT , and the initial detection test solution needs to be concentrated before detection; if When A _a -A _OPT > A _ss , the series N configuration of the interpenetration zone of the test solution is insufficient, and the series needs to be increased step by step to test again until A _a -A _OPT ≤ A _ss , A _ss is the computer preset The minimum allowable value of the absorbance detection error;

4) The computer calculates the arc length L ₀ between the laser measurement positioning hole and the average position of the absorbance value closest to the optimum absorbance value A _OPT according to the formula L ₀ = t _a × L / T ₁ , and then according to the formula C _x = C ₀ × L / L ₀ Calculate the concentration of C _x in the liquid to be tested, where, C ₀ =L _h / 0.434 α , L _h is the optical path length of the laser through the rotating disk microfluidic chip, and α is the absorption coefficient , L is the arc length of the mixed test solution measuring tape.

Compared with existing methods and technologies, the present invention has the following advantages:

(1) The present invention adopts a novel rotating disc microfluidic chip combined with the rotating dilution positioning technology to realize the effective positioning of the dilution concentration, thereby quickly calculating the dilution ratio, and multiplying the optimal absorbance position with the least noise by The method of dilution ratio is used to reverse the concentration of the liquid to be tested, which effectively reduces the detection error caused by the fixed optical path and makes up for the detection accuracy defect caused by the fixed optical path.

(2) The device is based on the rotary centrifugal sampling technology, without the need for additional sampling pumps and complicated sampling equipment, the sampling and detection devices are completed by a set of structures, and the sampling, detection and concentration calculation processes are all automatically controlled by computer Complete, high degree of automation.

(3) The solution concentration measuring device of the present invention has the function of automatic equal-proportional dilution, and the circular disc structure can increase the number of dilutions, thereby ensuring a higher dilution uniformity.

(4) The solution concentration measuring device of the present invention, the microfluidic chip adopts a circular disc structure, so that the sample injection and detection are carried out in the rotation mode, and the uniformity of the working mode ensures the uniformity of the sample injection and detection devices , so that the structure of the whole device is simple.

(5) The solution concentration measurement device of the present invention requires relatively common sample injection and detection equipment, is easy to be portable and commercialized, and can be used in all microfluidic photometric detection, with strong universality.

Description of drawings

Fig. 1 is a structural schematic diagram of a rotating disc microfluidic concentration measuring device based on photometric detection in the present invention;

FIG. 2 is an enlarged top view of the structure of the rotating disk microfluidic chip 1 in FIG. 1;

FIG. 3 is an enlarged schematic diagram of the connection structure between the rotating disc microfluidic chip 1 and the AC servo motor 13 in FIG. 1;

Fig. 4 is the left view of Fig. 3;

Fig. 5 is a schematic diagram of the support and connection structure for installing photomultiplier tube 18 and laser light source 23 in Fig. 1;

Fig. 6 is a specific working flow chart of a method for measuring the concentration of a rotating disk microfluidic solution based on photometric detection in the present invention.

The serial numbers and names of the components in the attached drawings: 1. Rotating disc microfluidic chip; 2. Test solution channel; 3. Original test solution pool; 4. Mixed test solution measuring belt; 5. Rotating disc microfluidic chip center Hole; 6. Test solution interpenetration zone; 7. Rotating disc microfluidic chip fixing groove; 8. Laser measurement positioning hole; 9. Microfluidic chip waste pool; 10. Shaft fixed hex nut; 11. Chip Fixed gasket; 12. Motor shaft positioning and fixing sleeve; 13. AC servo motor; 14. Motor shaft; 15. Microfluidic chip positioning key; 16. Photometric detection device bracket; ; 18. Photomultiplier tube; 19. Wire; 20. Motor control line; 21. Servo motor fixed base; 22. Detection device base; 23. Laser light source; 24, 29. Bracket loading arm; 26. Adjustable telescopic connecting arm; 27. Laser light source installation hole; 28. Fixed threaded hole; 31. Adjustable telescopic photomultiplier tube connecting arm; 32. Photomultiplier tube fixed threaded hole; 33. Motor control module; 34. Light source Control module; 35. Signal conversion module; 36. Computer; 37. Sealed dark room.

detailed description

Referring to FIG. 1 , it is an overall structural diagram of a rotating disk microfluidic concentration measuring device based on photometric detection according to the present invention. Inside a sealed darkroom 37, components such as a rotating disk microfluidic chip 1, an experimental device base 22, a photometric detection device bracket 16, and an AC servo motor 13 are arranged. Wherein, the experimental device base 22 is fixed on the bottom wall of the sealed darkroom 37, the photometric detection device support 16 is perpendicular to the experimental device base 22, and is also perpendicular to the bottom surface of the sealed darkroom 37, and the bottom of the photometric detection device support 16 is vertically welded and fixed In the center of the experimental device base 22 and the sealed dark room 37 near the left edge. The base 22 of the experimental device and the bracket 16 of the photometric detection device constitute the most basic supporting frame of the whole device. The servo motor fixed base 21 is fixed on the center of the experimental device base 22 and the sealed darkroom 37 near the right edge, the AC servo motor 13 is fixed on the center of the motor base 21, the output shaft motor shaft 14 of the AC servo motor 13 is vertically upward, and the motor The center of the shaft 14 is aligned with the center of the support 16 of the photometric detection device, and a horizontal rotating disc microfluidic chip 1 is coaxially installed on the motor shaft 14 . Two horizontal support loading arms 24, 29 are respectively fixedly connected to the photometric detection device support 16 by two positioning and fixing knobs 17, and the support loading arms 24, 29 are all horizontal to the right along the lateral center line of the left and right direction of the experimental device base 22 stick out. A laser light source 23 is installed on the right end of the support loading arm 24, so that the support loading arm 24 and the laser light source 23 are above the rotating disc microfluidic chip 1, parallel to the rotating disc microfluidic chip 1; on the support loading arm 29 The right end of the photomultiplier tube 18 is installed, so that the bracket loading arm 29 and the photomultiplier tube 18 are under the rotating disc microfluidic chip 1, and are also parallel to the rotating disc microfluidic chip 1. By adjusting the two positioning and fixing knobs 17, the two support loading arms 24, 29 can be moved along the photometric detection device support 16 to adjust the vertical height. The laser light source 23 and the photomultiplier tube 18 are respectively connected to the light source control module 34 positioned outside the sealed darkroom 37 by wires 19, the light source control module 34 is connected to the signal conversion module 35, and the signal conversion module 35 is connected to the computer 36; the AC servo motor 13 passes The motor control line 20 is connected to a motor control module 33 located outside the sealed dark room 37 , and the motor control module 33 is connected to a computer 36 .

Referring to Fig. 1 and Fig. 2, the rotating disk microfluidic chip 1 is a key component of the present invention, and the thickness of the chip is certain. Assuming that the thickness is 5mm, the optical path length of the laser passing through the chip is L _h = 5mm, Therefore, it can be known that the solution concentration C ₀ corresponding to the optimal absorbance position is constant , that is, C ₀ =L _h / 0.434 α , and α is the absorbance coefficient. The center hole 5 of the rotating disk microfluidic chip 1 is located at the center of the chip, and there is a chip fixing groove 7 at the center hole 5. The chip fixing groove 7 is a rectangular groove, and the center of the chip fixing groove 7 and the center The center of the hole 5 is consistent, the width of the chip fixing groove 7 is equal to the diameter of the central hole 5, and the length of the chip fixing groove 7 is about 3 times of its width. Next to the center hole 5 of the chip and the two sides of the chip fixing groove 7 and close to the center hole 5 and the chip fixing groove 7, an original test solution pool 3 is respectively set, and the two original test solution pools 3 have the same specifications, both of which are cube. The centers of the two original test solution pools 3 are both on the horizontal central axis in the front-back direction of the rotating disk microfluidic chip 1 , and are symmetrical with respect to the center hole 5 .

An N-level test solution interpenetration zone 6 is provided on the rotating disc microfluidic chip 1, and 3≤N≤100. Each test solution interpenetration zone 6 is in the shape of an arc with the center of the rotating disc microfluidic chip 1 as the center, and the test solution interpenetration zones 6 are arranged equidistantly along the radial direction of the rotating disc microfluidic chip 1 .

The two original test solution pools 3 are respectively connected to the innermost first-stage test solution interpenetration zone 6 through a small straight channel, and the two direct channels are parallel to each other and relative to the left and right direction of the rotating disc microfluidic chip 1 The transverse horizontal central axis is symmetrical. Because the test solution flowing through these two short straight passages has not been mixed, it is not necessary to make a spring like the spring-shaped test solution flow channel 2 to promote the mixing of the two test solutions. The midpoint of the first-stage test solution interpenetration zone 6 intersects the horizontal horizontal central axis of the rotating disc microfluidic chip 1 in the left and right directions at point B, and the two ends of the first-stage test solution interpenetration zone 6 are A and B respectively. point, the two straight channels are respectively connected to the midpoints of the AB and BC arcs of the first-stage test solution interpenetration zone 6 . The two ends of A, C and the middle point B of the first-stage test solution interpenetration zone 6 are respectively connected to the second-stage test solution interpenetration zone 6 through a spring-shaped test solution flow channel 2. These three spring-shaped test solution flow channels 2 The connection point with the interpenetration zone 6 of the second test solution divides the interpenetration zone 6 of the second test solution into four equal parts. From the two ends of the second-level test solution interpenetration zone 6 and the central point of the connection point of two adjacent spring-shaped test solution flow channels 2 on it, respectively connect the third-level test solution through a spring-shaped test solution flow channel 2 Interpenetration zone 6, like this, four spring-shaped test solution flow paths 2 are connected between the second-stage test solution interpenetration zone 6 and the third-level test solution interpenetration zone 6, and these four spring-like test solution flow paths 2 will The third test solution interpenetration zone 6 is divided into five equal parts. From the two ends of the third-level test solution interpenetration zone 6 and the central point of the connection point of two adjacent spring-shaped test solution flow channels 2 on it, respectively connect the fourth-level test solution through a spring-shaped test solution flow channel 2 The interpenetration zone 6, five spring-like test solution passages 2 are connected between the third-level test solution interpenetration zone 6 and the fourth-level test solution interpenetration zone 6, and these five spring-like test solution flow passages 2 connect the fourth Level test solution interpenetration zone 6 is divided into six equal parts. According to this rule, the test solution interpenetration zone 6 of the next stage is connected to the spring-shaped test solution flow channel 2 in turn until it is connected to the test solution interpenetration zone 6 of the last stage. The last level of test solution interpenetration zone 6 is called the mixed test solution measurement zone 4, and between the two ends of the mixed test solution measurement zone 4 is a laser measurement positioning hole 8, and the laser measurement positioning hole 8 is a through hole through the upper and lower sides. The two ends of the mixed test solution measuring tape 4 are as close as possible to but not in contact with the laser measurement positioning hole 8 , and the center of the laser measurement positioning hole 8 is located on the horizontal central axis in the left and right direction of the central hole 5 . The laser measurement positioning hole 8 is a rectangular slit left between the two ends of the mixed test solution measurement belt 4. The radial length of the laser measurement positioning hole 8 is larger than the radial width of the test solution measurement belt 4, but the laser measurement The width of the positioning hole 8 is very small. The laser light source 23 is located directly above the laser measurement positioning hole 8 , and the photomultiplier tube 18 is located directly below the laser measurement positioning hole 8 . The transverse horizontal central axis symmetry in the left and right directions means that the central diameter axis passes through the central hole 5 of the rotating disc microfluidic chip 1 and the central diameter axis passing through the laser measurement positioning hole 8 at the same time. The central hole 5 of the microfluidic chip 1 is symmetrical to the transverse central axis passing through the center of the laser measurement positioning hole 8 in the left-right direction.

The fan angle corresponding to each level of test solution interpenetration zone 6 is greater than that of the previous level of test solution interpenetration zone 6 degrees, of which Indicates the fan angle of the mixed test solution measurement zone 4, Indicates the fan angle corresponding to the interpenetration zone 6 of the first test solution, And between 30°~60°.

In order to facilitate subsequent detection, the mixed test solution measuring zone 4 is wider than the test solution interpenetration zone 6 of the previous stage, and the outside of the mixed test solution measuring zone 4 is connected to N+2 waste liquid pools through flow channels according to the previous equal division relationship 9. N+2 waste liquid pools 9 are evenly arranged along the arc of the mixed test solution measuring belt 4 , and two of the waste liquid pools 9 are respectively connected to both ends of the mixed test solution measuring belt 4 .

The rotating disk microfluidic chip 1 is made of PDMS (polydimethylsiloxane) material with uniform texture and good light transmittance. It is well fixed on the motor shaft 14 through its central hole 5 and the chip fixing groove 7; the unknown concentration solution and the diluent to be tested are respectively injected into the two original test solution pools 3, and when the rotating disc microfluidic chip 1 rotates around the center at a uniform speed After the rotation, the two test solutions in the two original test solution pools 3 will first pass through the spring-shaped test solution flow channel 2 and the test solution interpenetration belt 6 at all levels under the action of centrifugal force to the rotating disc microfluidic chip 1 slowly. because of the concentration difference between the two test solutions, the two test solutions are in the interpenetration zone 6 of each test solution (the number of stages of the interpenetration zone 6 of the test solution can be increased according to the gradient change of the concentration) When the test solution finally enters the mixed test solution measurement zone 4 for a period of time, a concentration gradient ring with uniform concentration changes will be formed in the test solution interpenetration zone 6 and the mixed test solution measurement zone 4. The test solution measurement zone 4 provides a fixed detection optical path for detection, and the concentration gradient ring changes most uniformly, with the highest resolution, and is most suitable for absorption photometric detection.

Refer to the connection structure between the rotating disk microfluidic chip 1 and the AC servo motor 13 shown in FIG. 3 and FIG. 4 . The rotating disc microfluidic chip 1 cooperates with the positioning key 15 through its central hole 5 and the chip fixing groove 7, so that the motor shaft 14 is firmly connected with the rotating disc microfluidic chip 1 . The use of the hexagonal nut 10 and the washer 11 to install on the motor shaft 14 can further improve the connection effect between the rotating disc microfluidic chip 1 and the motor shaft 14 . The positioning sleeve 12 is fixedly installed on the motor shaft 14 between the motor main body and the rotating disc microfluidic chip 1, so that the distance between the rotating disc microfluidic chip 1 and the motor main body can be determined, so that the rotating disc microfluidic chip 1 can be horizontally and stably fixed on the motor shaft 14 for rotation.

Participate in the supporting and connecting structures of the photomultiplier tube 18 and the laser light source 23 shown in FIG. 5 . The bracket loading arm 24 can be effectively fixed on the photometric detection device bracket 16 by means of the positioning and fixing knob 17 , and is perpendicular to the photometric detection device bracket 16 . The bracket loading arm 24 is a hollow tube, and a telescopic connecting arm 26 for installing the laser light source 23 is sleeved therein. The telescopic connecting arm 26 determines the telescopic length and fixes it by the fixing screw 25 . The laser light source 23 is fixed on the position directly above the mixed test solution measurement belt 4 through the laser light source installation hole 27 and the fixed threaded hole 28 at the right end of the telescopic connecting arm 26, so that the laser light source 23 emits a laser beam. In the same way, the support loading arm 29 determines the telescopic length and the fixed threaded hole 32 of the photomultiplier tube at its end through the adjustable telescopic photomultiplier tube connecting arm 31 to firmly fix the photomultiplier tube 18 directly below the mixed test solution measurement belt 4, The photomultiplier tube 18 is used to receive the light signal transmitted by the laser light source 23 .

Referring to Figures 1-6, when the photometric detection-based rotating disk microfluidic solution concentration measurement device of the present invention is working, the entire working process can be divided into the rotation speed adjustment stage, the test solution diffusion stabilization stage, and the photometric detection and judgment stage. details as follows:

Speed adjustment phase:

First prepare a diluent with a concentration of 0, inject it into one original test solution pool 3, and inject the test solution into the other original test solution pool 3, then set the AC servo on the computer 36 according to the difference of the test solution. The target speed V _set of the motor 13; the computer 36 initializes the signal conversion module 35, and the signal conversion module 35 can convert the analog electrical signal into a high-precision digital signal and transmit it to the computer 36; then turn on the laser light source 23 through the light source control module 34, and then The AC servo motor 13 is controlled by the motor control module 33 to start rotating counterclockwise from a static state. In order to avoid the impact of excessive acceleration on the diffusion process of the two test solutions, the computer 36 controls the rotation acceleration of the AC servo motor 13 to be a slow acceleration adjustment process, so as to ensure that the lateral force of the test solution in the rotating disc microfluidic chip 1 is almost is zero; as the rotation speed of the rotating disk microfluidic chip 1 increases, the two different test solutions will be affected by the centrifugal force, and thus begin to flow out from the two original test solution pools 3, and in the first-stage test solution interpenetration zone Mixing is performed for the first time in 6; the test solution will be further mixed through the spring-shaped test solution flow channel 2 while mixing, and continue to flow into the second-stage test solution interpenetration zone 6 for interpenetration, and pass through the spring-shaped test solution flow again Channel 2 flows to the next test solution interpenetration zone 6. As time increases, the rotation speed of the rotating disk microfluidic chip 1 will be stable, and the flow and interpenetration of the test solution will also be in a stable state. . When the rotating disk microfluidic chip 1 rotates, each time the laser light source 23 directly irradiates the photomultiplier tube 18 through the laser measurement positioning hole 8, an optical pulse signal will be generated and converted into a digital pulse signal by the signal conversion module 35 for transmission. For the computer 36, the computer remembers the number of times C according to the number of pulse signals. Simultaneously, when the computer 36 drives the AC servo motor 13 to rotate from the motor control module 33, that is, when the detection process officially begins, it calls the timer in the computer 36 to start counting, and the light signal emitted by the laser light source 23 passes through the laser measurement positioning hole 8 directly. The number of times C irradiated on the photomultiplier tube 18 and the time t measured by the timer can calculate the real-time rotational speed V _now = C/t(rad/s) of the rotating disk microfluidic chip 1, and the computer 36 can calculate the real-time rotational speed V now = C/t(rad/s) according to the target rotational speed V _set and For the comparison of the real-time rotation speed V _now , the motor control module 33 is used to adjust the rotation speed of the AC servo motor 13 until the rotation speed of the rotating disk microfluidic chip 1 is stabilized at the target rotation speed V _set , and the rotation speed adjustment stage is completed.

Stable stage of test solution diffusion:

After the rotation speed of the rotating disk microfluidic chip 1 is stable, the rotation period of the rotating disk microfluidic chip 1 is T ₁ =2π/ V _set , and the laser detection rate is much higher than the rotation rate of the rotating disk microfluidic chip 1, so The computer 36 starts counting when the laser measurement positioning hole 8 just rotates to the laser light source 23. When the time reaches T ₁ /2, record the light intensity value I that the current signal conversion module 35 converts and delivers to the computer 36, and the computer 36 converts the obtained The difference between the light intensity value I and the preset original light intensity signal I ₀ in the computer 36, that is, A ₀ = I ₀ - I , then the mixed test solution measurement belt 4 and the rotating disc microfluidic chip 1 are rotated. When the disk-type microfluidic chip 1 rotates T ₁ /2, the absorbance A ₀ corresponding to the point directly opposite to the laser measurement positioning hole 8 , that is, the middle point of the arc of the mixed test solution measurement strip 4 . The original light intensity signal I0 is the light intensity signal of the laser light source 23 passing through the empty mixed test solution measurement zone 4 without test solution when there is no test solution in the rotating disk microfluidic chip ₁ . Afterwards, the computer 36 repeatedly detects and judges whether the value of the _absorbance A0 will change. If there is a change, then continue to wait and detect. The concentration diffusion process has been stabilized, so far the test solution diffusion stabilization stage is completed.

Photometric detection and judgment stage:

After the penetration of the test solution is stabilized, the computer 36 divides the period T1 into _n =T1 /Tt equal parts ( _t1 ~ _{tn )} , and Tt is the signal conversion module 35 that converts the light intensity signal and transmits it to the computer 36. The final calculation is converted to The time required for absorbance A , due to the high photoelectric conversion speed and computer calculation speed, the value of Tt is very small, and the whole sampling process can be regarded as continuous. When the laser measurement positioning hole 8 just rotates under the laser light source 23, the timing starts, and the computer 36 calculates the absorbance A obtained from the first moment to the nth moment in turn, and makes a difference with the optimal absorbance A _OPT successively, that is, A _OPT - A , record the absorbance value A d when the difference is the smallest and the relative time t d to obtain the absorbance value A _d in _the period _T 1 _, and take the average over multiple cycles to obtain the average absorbance value A _a and the average time t _a . The optimal absorbance A _OPT is a predetermined value in the computer 36, that is, 1/ln10.

1) If the maximum value A _max of the absorbance A in each cycle is less than A _OPT , the computer 36 prompts that the concentration of the mixed test solution is too low. At this time, the initial detection test solution needs to be concentrated before detection. The difference between the maximum value A _max and the optimal absorbance A _OPT is calculated by calculating the concentration factor, that is, Q=2× A _OPT / A _max , so that the detected optimal absorbance point appears on the arc of the mixed test solution measurement zone 4 midpoint position. Because the penetration effect of the test solution at the middle point of the mixed test solution measurement zone 4 is the best, the concentration gradient is small, and the precision is high, so the detection error is the smallest.

2) If A _a -A _OPT > A _ss occurs, where A _ss is the minimum allowable value of the absorbance detection error preset by the computer 36 . The computer 36 prompts that the series N configuration of the test solution interpenetration zone 6 is insufficient, and the required resolution has not been reached. It is necessary to gradually increase the number of stages of the selected rotating disk microfluidic chip to detect again until A _a -A _OPT ≤ A _ss .

After the average time t _a is obtained, the computer 36 calculates the arc length between the laser measurement positioning hole 8 and the measured average position of the absorbance value closest to the optimum absorbance value A _OPT according to the formula L ₀ = t _a × L / T ₁ L ₀ , where L is the arc length of the mixed test solution measurement zone 4, and T ₁ is the rotation period of the rotating disc microfluidic chip 1. The solution concentration C ₀ corresponding to the absorbance A _a is the known concentration value C ₀ =L _h / 0.434 α , where α is the absorption coefficient of the absorbing substance, and L _h is the optical path length of the laser light passing through the chip , so it can be calculated according to The proportional relationship between the arc length L ₀ and the arc length L of the mixed test solution measurement zone 4 determines the dilution factor n ₂ between the concentration C ₀ and the concentration C _x of the test solution, that is, C _x = C ₀ × n ₂ = C ₀ × L / L ₀ , so as to calculate the precise concentration of C _x of the liquid to be tested. The effective compensation of the detection error caused by the fixed optical path in the microfluidic system is realized.

Claims (1)

1. A rotating disc microfluidic concentration measuring method based on photometric detection, using a rotating disc microfluidic concentration measuring device based on photometric detection, the device has a sealed darkroom (37), and a rotating disc is set inside the sealed darkroom (37). Disk-type microfluidic chip (1), photometric detection device bracket (16) and AC servo motor (13), the bottom end of the photometric detection device bracket (16) is vertically fixed in the center of the sealed darkroom (37) near the left edge, and the AC servo motor The motor (13) is fixed at the center of the sealed chamber (37) near the right edge, the output shaft motor shaft (14) of the AC servo motor (13) is vertically upward and coaxially equipped with a horizontal rotating disc microfluidic chip (1 ), close to the center of the rotating disc microfluidic chip (1) and set up an original test solution pool (3) symmetrically with respect to the center, and the rotating disc microfluidic chip (1) is equipped with N-level test solution interpenetration Belt (6), 3≤N≤100, each level of test solution interpenetration zone (6) is an arc shape with the center of the rotating disc microfluidic chip (1) as the center, two original test solution pools (3 ) are respectively connected by a short straight channel and communicated with the innermost first-stage test solution interpenetration zone (6), and the spring-like test solution flow channel (2) between adjacent two test solution interpenetration zones (6) is connected And connected, the outermost and last stage test solution interpenetration zone (6) is the mixed test solution measurement zone 4 whose width is greater than the previous stage test solution interpenetration zone (6); between the two ends of the mixed test solution measurement zone (4) There is a laser measurement positioning hole (8) that penetrates up and down. The photometric detection device bracket (16) is connected to the laser light source (23) directly above the laser measurement positioning hole (8) through the right end of the first bracket loading arm (24), Connect the photomultiplier tube (18) directly below the laser measurement positioning hole (8) through the right end of the second bracket loading arm (29); the laser light source (23) and the photomultiplier tube (18) are respectively connected to the sealed The light source control module (34) outside the darkroom (37) is connected, and the light source control module (34) is connected to the computer (36) through the signal conversion module (35); the AC servo motor (13) is connected to the sealed The motor control module (33) outside the darkroom (37) is connected, and the motor control module (33) is connected with the computer (36), which is characterized in that the following steps are followed successively; 1) Inject the diluent with a concentration of 0 into one original test solution pool (3), inject the test solution into another original test solution pool (3), and the computer (36) controls the rotation of the rotating disc microfluidic chip (1) , two different test solutions flow out from the two original test solution pools (3) under centrifugal force, and are mixed in the test solution interpenetration zone (6) and the spring-like test solution flow channel (2) at all levels, and the computer (36) according to The number of times C of the optical signal emitted by the laser light source (23) directly irradiating the photomultiplier tube (18) through the laser measurement positioning hole (8) and the time t measured by the timer are used to calculate the real time of the rotating disk microfluidic chip (1). Rotation speed V _now = C/t , adjust the rotation speed of the AC servo motor (13) until the real-time rotation speed V _now stabilizes at the target speed V _set ; 2) When the time reaches T ₁ /2, record the light intensity value I delivered to the computer (36) by the current signal conversion module (35), and calculate the absorbance here A ₀ = I ₀ - I , I ₀ is the original light intensity signal , T ₁ is the rotation period of the rotating disk microfluidic chip (1), T ₁ =2π/ V _set , the computer (36) detects and judges whether the value of absorbance A ₀ has changed multiple times, and continues to detect if there is a change, If it is stable, the concentration diffusion of the test solution in the mixed test solution measurement zone (4) has been stabilized; 3) The computer (36) sequentially calculates the absorbance A obtained from the first moment to the nth moment, and successively makes a difference with the optimal absorbance A _OPT , that is, A _OPT - A , and minimizes the absorbance value A _d and the cycle T Record the relative time t _d at which the absorbance value A _d is obtained within ₁ , and obtain the average absorbance value A _a and the average time t _a by averaging multiple cycles. If the maximum value A _max of the absorbance A in each cycle T ₁ is less than At A _OPT , the concentration of the mixed test solution is too low, and the concentration factor Q=2× A _OPT / A _max is calculated according to the difference between the maximum absorbance A _max and the optimum absorbance A _OPT , and the initial detection test solution needs to be concentrated before Detection; if A _a -A _OPT > A _ss , the series N configuration of the test solution interpenetration zone (6) is insufficient, and the series needs to be increased step by step to test again until A _a -A _OPT ≤ A _ss , A _ss is the minimum allowable value of the absorbance detection error preset by the computer (36); 4) The computer (36) calculates the arc length L ₀ between the laser measurement positioning hole (8) and the average position of the absorbance value closest to the optimum absorbance value A _OPT according to the formula L ₀ = t _a × L / T ₁ , Then calculate the concentration of the test solution C _x according to the formula C _x = C ₀ × L / L ₀ , where, C ₀ = L _h / 0.434 α , L _h is the laser passing through the rotating disc microfluidic chip (1) The optical path length, α is the absorption coefficient, and L is the arc length of the mixed test solution measurement zone (4).